Quantitative comparison of spark plasma's influence on parameters affecting rejuvenation of the skin at two different voltages

doi:10.21203/rs.3.rs-3148170/v1

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Quantitative comparison of spark plasma's influence on parameters affecting rejuvenation of the skin at two different voltages

https://doi.org/10.21203/rs.3.rs-3148170/v1

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The skin is the body's largest organ and protects against mechanical, thermal, and physical harm. However, the skin's function and appearance decrease visibly with age due to repeated exposure to adverse environmental impacts such as UV rays and toxic substances and the development of oxidative stress in aging. During aging, these factors cause phenotypic changes in the skin, such as wrinkles, pigmentation, loss of elasticity, and moisture. SPARK plasma has been found in studies to preserve skin against aging effectively.

The effects of two separate spark plasma device modes with voltages of 3.44 and 4.88 kV on skin health, including pigmentation, skin firmness, elasticity, and moisture loss, were studied using the MPA9 multifunctional skin tester in this study. In addition, utilizing ultrasound, the effectiveness of two plasma modes on skin density and thickness was examined.

The findings of this study suggest that spark plasma may slow the skin's aging process by enhancing its elasticity, density, and thickness. However, there were no difficulties or scars at the treatment location at the end of this study, indicating that Spark plasma can be utilized as a new way to delay the aging process.

Health sciences/Medical research

Physical sciences/Engineering

Physical sciences/Physics

Cold plasma

Spark

rejuvenation

Skin

Skin rejuvenation is one of the most popular cosmetic treatments in Western countries to reduce age-related changes like wrinkles and uneven pigmentation [1], in which plasma can be an effective tool[2]. Plasma is the fourth state of matter, which can be created by applying high voltages to gaseous elements or compounds [3]. Plasma includes ions, free electrons, free radicals, and reactive oxygen and nitrogen species which constitutes a medium that can be used in medical applications and is the basis of the so-called science “plasma medicine.” Plasma medicine is an emerging interdisciplinary field of research with rapidly growing importance, exploring considerable opportunities at the interface of plasma physics with life sciences, medicine, and engineering [3]. The plasma application in clinical medicine was first reported at the beginning of the 20th century when Jacques-Arsène d’Arsonval, The French physiologist, invented his medical device known as “Arsonvalization.” [4]

The skin is the body's outermost layer and, with an extensive surface area of approximately 2 m2 and weighing about 3kg, forms the first line of defense against various external pathogens and environmental influences. The skin's structure and cellular composition work harmoniously to prevent infection and deal with physical and chemical challenges from the outside world [5]. The skin organ consists of three tissue layers: from the surface inwards are the epidermis, dermis, and hypodermis. The epidermis consists mainly of epithelial cells, known as keratinocytes, which are protein-rich epithelial cells with abundant keratin and keratohyalin. The dermis is a connective tissue with fibroblast cells interspersed in a collagen-elastin-based extracellular matrix. This layer also includes blood vessels, lymph, sweat glands, nerve endings, and hair follicles [6], [7]. At the macroscopic level, the most critical biophysical properties of the skin are water content, thickness, transepidermal water loss (TEWL), sebum production, surface pH, and melanin index.[8]

Two main types of cells make up the skin epidermis: the keratinocytes, the most abundant cells that are present in all layers of the epidermis and produce keratin to protect the epithelial cells from mechanical and non-mechanical stress, and melanocytes, which are present in the basal layer of the epidermis and produce the protective pigment melanin. [9] Skin pigmentation results from three distinct processes: (i) melanin biogenesis and transport within melanocytes; (ii) melanin transfer from melanocytes to keratinocytes; and (iii) melanin uptake and processing by keratinocytes. Within the keratinocytes, melanin accumulates in the supranuclear region and protects the genetic core material from UVR-induced damage. [10]

Transepidermal water loss is the flow of water that leaves the body through the skin. It is the most commonly used parameter to assess skin barrier function and a good indicator of pathological conditions such as irritation, atopic eczema, contact dermatitis, and psoriasis [11], [12]. Collagen fibers have high tensile strength and ensure the skin’s stability, while elastin is responsible for elasticity and flexibility. The first step of aging happens when elastin does not play its role, and the loosening process of the skin begins. Signs of aging usually include wrinkling, rough texture, sallow complexion with mottled pigmentation, and loss of skin elasticity. [13] In the past, the effect of plasma jet utilizing argon as a feed gas on mentioned skin properties has been reported. It can be seen that the water content in the SC (stratum corneum) is reduced after plasma treatment, particularly in the upper skin layers.[14] laser confocal microscopy also shows improvement in collagen structure that has changed shape due to aging, which helps the skin regain strength and elasticity. [15]

This study sought to determine the quantitative effects of spark plasma at two different voltages on various skin properties, including skin elasticity and strength, layer thickness and density, melanin index, erythema index, stratum corneum hydration, and epidermal water loss. For the purpose of comparing the benefits and drawbacks of each of these voltages, all the aforementioned parameters were quantitatively and statistically examined.

Visual assessment of plasma performance

Figure 1 depicts the changes in the skin's look over four weeks following a plasma treatment session. The skin appearance changes following a 4.88 kV plasma treatment session are shown in the first row, and the changes following a 3.44 kV plasma treatment session are shown in the second row.The photos in Fig. 1 show significant changes in the skin after treatment. The immediate effect of plasma on animal samples was significant. It was decided to use quantitative tests to assess effectiveness for a more thorough evaluation. It's worth noting that the burned areas vanished after two weeks, leaving no visible scars. The shortening of the surface hair of the skin causes small red spots. All minor and transient side effects subsided within two weeks. Figures (1, b) also shows skin contraction from 4.70 mm to 3.70 mm (21.27% reduction) at 4.88 kV voltage and from 3.90 mm to 3.30 mm (15.38% reduction) at 3.44 kV after plasma therapy, demonstrating that spark plasma can be used for both skin lifting and wrinkle elimination.

Effects of Plasma on biometric parameters

Cutometer. According to Table 1, the average value of the parameter R2, which indicates the resistance to mechanical force versus the ability to recover, at the voltage of 3.44 kV before processing was 0.5467, but it increased to 0.6173 immediately after processing, which is most likely due to skin shrinkage. It climbed dramatically after two weeks, reaching a value of 0.6929, and finally maintained this linear development in the fourth week of follow-up. It had a value of 0.7458 compared to before therapy (a 36.41% rise). At a voltage of 4.88 kV, the average R2 was 0.5661 before processing, but it climbed to 0.6369 immediately. It rose after two weeks compared to before the treatment, reaching a value of 0.6459, and eventually maintained this linear growth in the fourth week of follow-up. Its value in comparison to before treatment was 0.7682 (an increase of 35.7%). This growth trend at both voltages suggests skin tolerance increases after plasma therapy. (Table 1)

Furthermore, as shown in Table 1, after one session of plasma processing, the average value of R5, which depicts the elastic part of the suction phase versus the immediate recovery in the relaxation phase, steadily rose. At a voltage of 3.44 kV, the average value immediately after treatment increased from 0.4627 to 0.6596. It grew considerably in the second week of tracking, reaching an average of 0.6890. Furthermore, it increased significantly in the fourth week of follow-up, reaching an average of 0.6235. However, at a voltage of 4.88 kV, the average value R5 immediately after treatment jumped from 0.4032 to 0.5936. It increased considerably in the second week of follow-up, reaching an average of 0.6768. It also averaged 0.6365 in the fourth week of follow-up. Additional proof in favor of the idea that plasma improves skin elasticity may be seen in Table 1.

Table 1

Comparison of skin elasticity parameters in rats after receiving plasma for four weeks [*p < 0.05, a significant difference from before the start of the experiment.]
Measurement time	Before	After procedure	After two weeks	After four weeks	P value	The percentage of changes
R2 (4.88kv)	0.5661	0.6369	0.6459	0.7682	0.0323 p < 0.05	35.7%
R5 (4.88kv)	0.4032	0.5936	0.6768	0.6365	0.0223 p < 0.05	57.86%
R2 (3.44kv)	0.5467	0.6173	0.6929	0.7458	0.0428 p < 0.05	36.41%
R5 (3.44kv)	0.4627	0.6596	0.6890	0.6235	0.0258 p < 0.05	34.75%

Tewameter. Table 2 demonstrates that the treatment groups' skin surface saw a considerable rise in evaporation rate (g/h/m2) due to heat transmission to the skin, indicating that the skin had significantly lost water. The average evaporation rate in the plasma group with 3.44 kV voltage went from 13.22 to 109, then reduced to 12.62 in the second week of follow-up. This indicates that the skin regeneration process is most likely complete. In the fourth week of the trial, the mean fell below the baseline and registered a value of 10.62. Also, in the plasma group with a voltage of 4.88 kV, the average evaporation rate climbed from 10.81 to 107.3, then reduced to 11.10 in the second week of follow-up before reaching 14.16 in the fourth week of the study. This demonstrates that plasma does not impact the skin barrier and does not cause the skin to dry out. Additionally, it meets the fundamental standards for protecting human skin.

Table 2

Changes in mouse skin's Tewameter parameter over four weeks following plasma processing
Measurement time	Before	After procedure	After two weeks	After four weeks	P value
3.44kv	13.22	109	12.62	10.62	0.5891 p > 0.05
4.88kv	10.81	107.3	11.10	14.16	0.1514 p > 0.05

Mexameter. The melanin level and the number of skin pigments at voltages of 3.44 and 4.88 kV have dramatically increased after the treatment, as shown in Fig. 2, from 75.28 to 121.5 and from 87.39 to 148.8, respectively. It is most likely caused by black soot from minor skin burns. In the fourth week of follow-up, the average melanin dropped to 79.67 and 101.4, respectively, and did not significantly vary from the baseline.

Also, as shown in Fig. 3, the quantity of erythema on the skin increased dramatically in the plasma group with a voltage of 3.44 kV, from 167.5 to 262.9 immediately after treatment, indicating inflammation. The amount of erythema increased from 155.3 to 265.6 in the plasma group with a voltage of 4.88 kV, showing that the plasma group with a voltage of 4.88 kV had a higher level of inflammation than the plasma group with a voltage of 3.44kv. However, the mean reduced to 148.3 in the 3.44 kV plasma group and 154.4 in the 4.88 kV plasma group in the second week of follow-up, showing a decrease in inflammation. After four weeks, they reached an average of 174.9 and 181.8, respectively, with no discernible change from pre-treatment levels. Figure 3 lends weight to the hypothesis that the plasma does not need to interact with the chromophore.

Skin ultrasonography. Figure 4 demonstrates that the average skin thickness in both groups of plasma with voltage 3.44Kv and plasma with voltage 4.88Kv fell immediately after treatment, from an average of 810.5 µm to 801 µm in group 3.44Kv and from an average of 744.5 µm to 731.3 µm in group 4.88Kv. However, once the skin healing process was completed, the average skin thickness in both groups grew in the second week. It reached a level higher than the average before treatment. This increase was maintained and substantially improved above the baseline level in the fourth week of follow-up. The average skin thickness in the plasma group with voltage 4.88Kv reached 990.7 µm, representing a 33.06% increase over the baseline, and 1019 µm in the plasma group with voltage 3.44Kv, representing a 25.72% increase.

The average skin density has significantly increased, as seen in Fig. 4. Average skin density changed by 46.43% in the plasma group with voltage 3.44Kv, but only by 18.24% in the plasma group with voltage 4.88Kv. The persistence of the rise in the average skin density raises the chance that significant alterations will be seen with extended follow-up.

Animal models and research groups

In this experiment, male Wistar rats (4 months old) weighing 250 + 50gr were obtained from the Pasteur Institute (Tehran, Iran). Twelve rats were divided into two groups and kept in separate cages with easy access to water and food under standard laboratory conditions (room temperature, atmospheric pressure, humidity 30 + 10%, light/dark cycle 12 hours). Following the experiment, all mice were donated to the Faculty of Biology at Shahid Beheshti University for survival. The Tehran University of Medical Sciences ethics committee

Plasma device

The spark plasma from Plasma Technology Jam (Plasma Beauty 100) was used in two modes in this work. Using an oscilloscope and a high voltage probe, the electrical properties of the plasma device in these two modes were investigated and demonstrated (Tekterorix, P6015A, 1:1000). The voltage variations are sinusoidal, as illustrated in Figure (5). n medium mode, this device has a peak-to-peak voltage of 3.44 kV and a frequency of 62.5 kHz, whereas in high mode, the peak-to-peak voltage is 4.88 kV, and the frequency is 71.09 kHz.

The numerous active species of cold plasma were investigated using optical emission spectroscopy (OES; Avaspec3648USB2). Along the plasma axis, OES spectra were taken in the 250–800 nm region. Optical emission spectroscopy of the plasma device showed the emission of species such as OH (309 nm), NO (297 nm), and N2/N2+ (315, 337, 358, 375.4, and 380 nm)[16] in Figure (6).

Biometrics evaluation

Using a multipurpose skin tester (Courage + Khazaka Electronics, Cologne, Germany), the samples' biometric properties—including melanin index, erythema index, stratum corneum hydration, and epidermal water loss—were assessed. A Dual Cutometer MPA 580 from Courage + Khazaka Electronic GmbH (Köln, Germany) was used to calculate the elasticity index.

The thickness of the skin layers was measured using a skin ultrasonic imaging device (Dub®SkinScanner Taberna pro medium, Luneburg, GERMANY). Structures can be seen using this instrument up to a maximum depth of 1 cm. In this experiment, a probe with a frequency of 75 MHz was used. Biometric analysis of skin samples was done before and after treatment at 2 and 4 weeks. All measures were acquired while relaxing in a controlled physical setting (23 ° C room temperature and 40% humidity).

Treatment and preparation

To assess the efficacy of SPARK plasma and any potential impacts on skin renewal in two modes, 12 Wistar rats were split into two groups of six. Plasma delivered to the first group had a voltage of 3.44 kV, while plasma delivered to the second group had a voltage of 4.88 kV.

Before plasma treatment, rats were anesthetized with ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (10 mg/kg). The area behind the rats' necks was shaved and measured with a ruler after being trimmed short with scissors. Before irradiation, the mouse's skin was cleansed, and a topical Xyla-P cream was used to give a local anesthetic. After 20 minutes, the required region was cleaned and treated in two modes utilizing the SparkMED (Fig. 7) device in a triangle pattern. After the procedure, the treated area's size was measured more with a ruler, and the quantity of skin shrinking was also determined. Skin and biometric analysis were performed before and immediately after the treatment and in the second and fourth weeks to report potential side effects of this procedure and compare the two modes.

Statistical analysis

Results were expressed as mean ± standard error of the mean (mean ± SEM). Statistical data analysis was performed by applying way-ANOVA to compare the groups using the Graph Pad Prism (9.0.0) software. The significance level was considered less than 0.05 (p < 0.05).

As the world population ages, people from all walks of life desire to maintain a healthy lifestyle and appear younger[17]. Clinical signs of skin aging, which include intrinsic and photoaging, include wrinkles, pigmentary changes, skin thinning, and telangiectasias[18]. Age-related changes in the skin's structural stability and physiological function are caused by a combination of factors, including genetics, a drop in estrogen levels, exposure to UV radiation from the sun, and changes in estrogen levels[19]. The accumulation of reactive oxygen species (ROS), a by-product of cellular metabolism, which causes damage to important cellular components like deoxyribonucleic acid (DNA), enzymes, and membranes, causes intrinsic aging, which is also influenced by an individual's genetic makeup and occurs at varying rates in all organisms[18]. On the other hand, skin rejuvenation is a popular cosmetic procedure for removing age-related changes such as wrinkles and uneven pigmentation [1].

Many therapeutic strategies have been investigated to achieve an acceptable outcome with no or few side effects. Laser therapy is a popular option among several medical and surgical therapies. Although ablative laser resurfacing is the gold standard for skin rejuvenation, treatment is typically linked with a lengthy recovery period and negative side effects.[20]

Using a new laser treatment technique called fractional photothermolysis, several microscopic thermal injury zones are produced with precisely controlled breadth, depth, and density and are encircled by a reserve of epidermal and dermal tissue, enabling quick healing of the laser-induced thermal injury. Fractionated carbon dioxide (CO2) laser resurfacing, in particular, combines the concept of fractional photothermolysis with the thermal effects associated with the 10,600-nm wavelength.[21] Compared to standard CO2 laser resurfacing, this technique effectively treats rhytides, skin laxity, and photodamaged skin with a shorter recovery period and fewer side effects.[21],[20]

The search for substitute techniques and apparatus that might use processes other than laser technology has been prompted by the desire to get beyond the constraints and challenges given by lasers. According to Shakouri et al., utilizing a portable plasma device in conjunction with vitamin C ointment has a good impact on parameters that are useful for skin rejuvenation.[16] Ghasemi et colleagues discovered that a plasma spark therapy session increases skin elasticity greatly, and ultrasound results demonstrated a significantly enhanced skin thickness and density.[22]

SparkMed's medical device produces plasma energy by ionizing the air near the skin. The ensuing plasma spark sublimates the surface layers, quickly sends the stored heat energy to the skin surface, and uniformly heats it.[23][24] P-RF (Plasma radiofrequency) energy generates microplasma sparks in the air between the device's tip and the skin's surface, causing mild epidermal erosion and superficial perforation of the skin with a 1 mm diameter spot. Mild epidermal sublimation protects the epidermis and avoids harm to the skin's deeper layers. In addition to a mechanical effect that shapes the surface on which it impacts, the detached spots of the sublimation technique induce a thermal effect that promotes skin regeneration and extensive dermal fibroblast remodeling, including new collagen synthesis and deposition, while also stimulating rapid re-epithelialization[25]. Heat shocks can promote the expression of procollagen type I and procollagen type II [25]. As a result, they can trigger cells to make more collagen. However, studies show that NO significantly impacts the skin, specifically on the proliferation of keratinocytes and fibroblasts [16], [22], [26] Epidermis thickening brought on by cold plasma is most likely caused by the proliferation of keratinocytes [16], [22], [26]. However, CAP can initially weaken the skin barrier and dry up the top layer of the epidermis. The human stratum corneum experienced a temporary in vivo water loss after plasma exposure [14], [22]. The non-dried epidermis shields thermally injured layers during recovery, a promising result of plasma skin resurfacing [22], [27]. Because CAPs can deposit charges on the treated surface, plasma treatment may cause the skin to absorb more water molecules[28]. As a result, various cold plasma-related physical elements can influence cellular activity and the skin's microenvironment, individually or in combination[17]. All of this highlights the beneficial role of CAP in skin biology and provides compelling arguments in favor of employing cold plasma to repair the skin's functional barrier and improve its health. Furthermore, the plasma does not need to interact with the chromophore[29][16]. Consequently, it generates more reliable outcomes than ablative lasers [16], [22], [27].

According to the findings of this study and in accordance with earlier studies[16], [22], one benefit of this method is the quick healing of the wound surface, the quick formation of the protective layer after treatment, the quick return to normal activity, and the best results in increasing skin elasticity.

Skin rejuvenation with plasma is one of the latest treatments that can stimulate skin regeneration with a short recovery period and without severe side effects or tissue damage. [16], [22]This study found that a plasma device can improve skin rejuvenation by boosting skin thickness, density, and flexibility. Furthermore, we discovered in this study that plasma with a voltage of 4.88Kv causes more changes than plasma with a voltage of 3.44Kv, with the exception that the recovery period of plasma with a voltage 4.88Kv may be more extended than that of plasma with a voltage 3.44Kv.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Animal ethics and ARRIVE guidelines

Animal experiments were conducted according to the Guidelines for Animal Care and Use Committee of Tehran University of Medical Sciences. All animal experiments described were approved by the Animal Experimentation Ethics Committee of Tehran University of Medical Sciences, IRAN (IR.TUMS.MEDICINE.REC.1400.766). The in-vivo study is reported in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) for reporting experiments [32-33].

Funding information:

Skin and Stem Cell Research Center,

Grant/Award Number: 1400-2-200-54971

Tehran University of Medical Sciences

Plasma Medicine Lab, Laser and plasma research Institute, Shahid Beheshti University

Author contributions

E.G, M.A.N, and M.R.K. initiated the research and designed the experiments. M.A.N, M.A.A, B.S, and M.R.K. conducted all parts of the experiments. E.G, M.N, and P.C. performed biological analyses, and E.G, P.C, and M.E carried out plasma treatment and characterization and analyzed the data. E.G and M.R.K and M.A.N wrote the manuscript. All authors discussed the result and revised the manuscript.

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No competing interests reported.

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Quantitative comparison of spark plasma's influence on parameters affecting rejuvenation of the skin at two different voltages

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Abstract

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Introduction

Result

Visual assessment of plasma performance

Effects of Plasma on biometric parameters

Materials and methods

Animal models and research groups

Plasma device

Biometrics evaluation

Treatment and preparation

Statistical analysis

Discussion

Conclusion

Declarations

References

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